KR20100110525A - Cryopreservation methods for chrysanthemum shoot tips - Google Patents
Cryopreservation methods for chrysanthemum shoot tips Download PDFInfo
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- KR20100110525A KR20100110525A KR1020090028899A KR20090028899A KR20100110525A KR 20100110525 A KR20100110525 A KR 20100110525A KR 1020090028899 A KR1020090028899 A KR 1020090028899A KR 20090028899 A KR20090028899 A KR 20090028899A KR 20100110525 A KR20100110525 A KR 20100110525A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/02—Dehydrating; Subsequent reconstitution
- A23B7/024—Freeze-drying, i.e. cryodessication or lyophilisation
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/04—Freezing; Subsequent thawing; Cooling
- A23B7/045—Thawing subsequent to freezing
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/04—Freezing; Subsequent thawing; Cooling
- A23B7/05—Freezing; Subsequent thawing; Cooling with addition of chemicals or treatment with chemicals other than cryogenics, before or during cooling, e.g. in the form of an ice coating or frozen block
- A23B7/055—Freezing; Subsequent thawing; Cooling with addition of chemicals or treatment with chemicals other than cryogenics, before or during cooling, e.g. in the form of an ice coating or frozen block with direct contact between the food and the chemical, e.g. liquid nitrogen, at cryogenic temperature
Abstract
Description
본 발명은 작은 방울-유리화법에 기초한 국화 신초의 초저온 동결보존방법에 관한 것이다. 좀 더 구체적으로, 본 발명은 액체 질소를 이용하여, 국화 및 국화와 같은 숙근 영양체 번식자원의 식물조직을 동결보존하는 방법에 관한 것으로서 전배양 과정, 로딩 및 탈수과정, 동결 및 해동과정, 재생 배양을 선별하여 국화 신초를 액체 질소에 동결보존하는 방법에 관한 것이다.The present invention relates to a cryogenic cryopreservation method of chrysanthemum shoots based on the droplet-vitrification method. More specifically, the present invention relates to a method of cryopreserving the plant tissues of root nutrient propagation resources, such as chrysanthemum and chrysanthemum, using liquid nitrogen, which includes preculture, loading and dehydration, freezing and thawing, and regeneration. The present invention relates to a method of cryopreserving chrysanthemum shoots in liquid nitrogen by selecting a culture.
마늘, 국화 등 이른바 영양체는 씨앗으로 번식하지 않고 영양체로 번식하기 때문에 저장고에서 장기저장을 할 수 없고 매작기마다 포장에 심어서 수확 저장 후 다시 심는 등 유지관리 비용이 많이 들고 생리적·병리적 퇴화 등으로 인해 우량한 품종 등 유전자원이 소실되는 위험성이 상존하고 있다. 독일에서 마늘 유전자원의 포장 보존시 매년 1.5%의 자원이 소실되었다는 보고도 있다(Keller et al., 2008).The so-called nutrients such as garlic and chrysanthemum do not multiply as seeds, but because they are propagated as nutrients, they cannot be stored for a long time in the cellar. There is a risk that genetic resources such as superior varieties are lost. It is also reported that 1.5% of the resources are lost each year in the conservation of garlic genetic resources in Germany (Keller et al., 2008).
한편, 국화는 가장 오랜 재배역사를 가진 화훼 작물 중의 하나로서 현재도 유럽과 일본, 한국 등지에서 육종되고 널리 재배되고 있다. 국제 화훼시장에서 소비자들의 요구는 자주 급속하게 바뀌고 있고 신품종이 시장에 소개되면 오래된 품 종들은, 유행이 지난 품종들이 미래에 중요해지기도 함에도, 자주 급속하게 사라지게 된다. 따라서 영양체 유전자원의 유일한 장기보존방법으로 간주하고 있는 초저온 동결보존법의 개발 및 실용화는 육종가들 뿐만이 아니라 재배자에게도 변화하는 시장수요에 신속하게 대응하기 위한 중요한 수단이 될 수 있다.On the other hand, chrysanthemum is one of the longest flowering crops and is still grown and widely cultivated in Europe, Japan and Korea. Consumer demand in the international flower market is often changing rapidly, and when new varieties are introduced to the market, older varieties are often rapidly disappearing, even though fashionable varieties may become important in the future. Therefore, the development and practical use of cryogenic cryopreservation method, which is regarded as the only long-term preservation method of nutrient gene source, can be an important means to respond quickly to changing market demand not only for breeders but also for growers.
식물조직의 동결보존기술은 전통적인 방법인 완속동결(2단계 동결)법에 의해 현탁배양세포와 같이 미세한 세포주에만 이용되었으나, 유리화(vitrification)법이 시도되면서 (Fahy, Cryobiology, 1984) 주류를 이루게 되었고, 특히 신초와 같이 교적 큰 조직에서도 캘러스 형성 등 지체기를 거치지 않고 재생식물체를 획득할 수가 있게 되었다. 초저온동결보존법이 영양체나 난저장(recalcitrant) 종자의 장기보존을 위한 유일한 옵션으로 인식되어 왔고, 근래에 감자, 바나나 등 기내 유식물에서도 동결보존의 대규모 이행사례가 알려지고 있고 전 세계적으로 확대되고 있다.The cryopreservation technique of plant tissues was used only for fine cell lines such as suspension culture cells by slow freezing (two-step freezing) method, which became the mainstream with the attempt of vitrification (Fahy, Cryobiology, 1984). In addition, regenerated plants can be obtained without delay, such as callus formation, even in large gyoza such as shoots. Cryogenic preservation has been recognized as the only option for long-term preservation of nutrients and recalcitrant seeds. Recently, large-scale implementation of cryopreservation has been known and expanded globally in inflight seedlings such as potatoes and bananas. .
작은 방울-유리화법(droplet-vitrification method)은 기존에 알려진 작은 방울-동결법(droplet-freezing)과 용액에 기반을 둔 유리화(vitrification)법의 장점을 혼합한 형태의 기술로서 본 개발자들에 의해 감자, 마늘 등에서 개발된 바 있고, 세계적으로도 각광받고 있는 신기술이다. 이 보존법의 가장 큰 장점은 냉각 및 해동 속도를 향상시켜서 재생률을 높이는 데 있다고 할 수 있는데, 기존의 유리화법에서는 크라리어 바이알에 용액과 시료를 넣어서 냉각 및 해동시키는데 반하여, 이 방법에서는 탈수시킨 시료를 작은 알루미늄 호일 스트립 위에 얹어서 액체 질소에 직접 냉각시키고, 언로딩 용액에 직접 담가서 해동시킨다. 냉각 및 해동속도는 시료의 재생률을 좌우하는 주요 요인 중의 하나인데, 냉각과정에서 유리화가 불완전하게 일어날 경우에는 특히, 해동속도가 빨라야만 탈유리화(devitrification)를 막을 수 있다. 이를 위해 체적이 큰 크라이어 바이알 대신 가늘고 체적이 적은 스투로우, open-pulled straw, 그리드(grid) 등을 사용하게 되는데, 이들은 크기가 작은 시료에만 적용할 수 있고, 보존관리가 다소 불편하며 가격이 비싼 단점이 있다. 이에 비해 알루미늄 호일을 이용할 경우 쉽고 저렴하게 열전달을 빠르고 균일하게 할 수 있다.The droplet-vitrification method combines the advantages of the known droplet-freezing and solution-based vitrification methods. It has been developed in the field of garlic and garlic, and it is a new technology that is in the spotlight worldwide. The main advantage of this preservation method is to increase the regeneration rate by improving the cooling and thawing speed. In the conventional vitrification method, the solution and the sample are cooled and thawed by adding the solution and the sample to the carrier vial. It is placed on a small strip of aluminum foil, cooled directly with liquid nitrogen, immersed directly in the unloading solution and thawed. The rate of cooling and thawing is one of the major factors in determining the regeneration rate of the sample. In the case of incomplete vitrification during the cooling process, defrosting can be prevented only when the thawing speed is high. For this purpose, instead of bulky cry vials, thin, low volume straws, open-pulled straws, grids, etc. are used, which can be applied only to small samples, which are somewhat inconvenient to maintain and costly. There is an expensive disadvantage. In contrast, when aluminum foil is used, heat transfer can be performed quickly and uniformly easily and inexpensively.
유리화법에 기반을 둔 초저온동결보존에 있어서 샘플 안에 있는 얼 수 있는 거의 모든 물을 제거하는 것이 필수요건이기 때문에 (Engelmann, 2000), 고농도의 동결보호제 혼합액인 유리화 용액에서의 탈수과정을 포함한다. 따라서 적절한 유리화 용액의 선정과 유리화 용액에서의 탈수시간이 동결보존한 마늘 신초의 재생에 가장 크게 영향 있는 요인이다 (Kim et al., 2006). 식물의 동결보존에 사용된 유리화 용액중 PVS2 (30% glycerol + 15% DMSO + 15% EG + 0.4M sucrose, Sakai et al., 1990)는 200종/품종의 신초에 성공적으로 적용되는 등 가장 널리 사용되어왔다 (SakaiA & Engelmann, 2007). PVS3 (Nishizawa et al., 1993). 또한 고추냉이(Matsumoto et al., 1995), 아스파라거스(Nishizawa et al., 1993) 및 마늘 (Kim et al., 2004) 등에 사용되었다. Since cryogenic preservation based on vitrification is a requirement to remove almost all of the frozen water in the sample (Engelmann, 2000), it involves dehydration in vitrification solutions, a high concentration of cryoprotectant mixture. Therefore, the selection of an appropriate vitrification solution and the dehydration time in the vitrification solution are the most influential factors for the regeneration of cryopreserved garlic shoots (Kim et al., 2006). PVS2 (30% glycerol + 15% DMSO + 15% EG + 0.4M sucrose, Sakai et al., 1990) in the vitrification solution used for cryopreservation of plants was most widely applied, including successfully applied to 200 varieties / cultivars of shoots. Has been used (SakaiA & Engelmann, 2007). PVS3 (Nishizawa et al., 1993). It was also used in horseradish (Matsumoto et al., 1995), asparagus (Nishizawa et al., 1993) and garlic (Kim et al., 2004).
Fahy et al. (Fahy et al., 2004)은 복잡하고 공간적으로 확장된 생물체계의 동결보존에 있어서 동결보호제의 독성이 가장 중요한 장벽이 되고 있다고 지적하였는데, 독성은 주로 침투성 동결보호제로 인해 발생한다고 알려져 있다(Fahy et al., 1984; Fahy et al., 1990). Fahy et al. (Fahy et al., 2004) pointed out that the toxicity of cryoprotectants is the most important barrier in cryopreservation of complex and spatially expanded biological systems. Toxicity is known to be caused primarily by invasive cryoprotectants (Fahy). et al., 1984; Fahy et al., 1990).
로딩 용액은 고농도의 유리화 용액에 접촉함으로써 받는 독성을 완화시키기 위해 시료를 유리화 용액보다 저농도의 동결보호제 혼합액에 배양할 때 사용하는 용액이다. 동결보호제 로딩 처리는 유리화 용액에서의 탈수과정에서 발생하는 장해를 최소화하는데 우선적으로 기여하는데, 세포막과 단백질의 탈수로 유도되는 불안정화를 방지한다. 또한, 로딩과정은 세포질의 용액농도를 증가시켜서 액체 질소에 담그는 동안 세포질을 유리화시키는데도 기여한다(Steponkus et al., 1992). Nishizawa 등(1993)도 로딩 처리가 동결-탈수에 대한 저항성 또는 탈수 저항성을 유도하는데 효과가 있다고 하였다. 짧은 로딩 처리시간 동안 세포는 상당한 탈수에 의해 원형질 분리가 일어나지만, 세포질 안으로는 글리세롤의 침투는 거의 일어나지 않았다 (Matsumoto 등, 1998). The loading solution is a solution used to incubate the sample in a lower concentration of cryoprotectant mixture than the vitrification solution to mitigate the toxicity received by contacting the high concentration vitrification solution. Lyoprotectant loading treatment primarily contributes to minimizing the disturbances that occur during dehydration in vitrified solutions, preventing destabilization induced by dehydration of cell membranes and proteins. The loading process also increases the solution concentration of the cytoplasm and contributes to the vitrification of the cytoplasm during immersion in liquid nitrogen (Steponkus et al., 1992). Nishizawa et al. (1993) also found that loading treatment was effective in inducing freeze-dehydration resistance or dehydration resistance. During the short loading treatment cells were proliferated in plasma by significant dehydration, but little penetration of glycerol into the cytoplasm (Matsumoto et al., 1998).
국화는 유전적 균일성을 고려해서 포장에서 영양체 자원으로 유지보존되고 있어서 생리적·병리적 퇴화 등으로 인한 소실이 우려되며, 많은 유지비용을 부담해야 한다. 이를 극복하기 위해 냉각속도 조절에 의한 slow-freezing (Fukai, 1990, 1991; Halmagyi et al., 2004), 알긴산 코딩 및 탈수처리에 의한 encapsulation-dehydration (Martin and Gonzalez-Benito, 2005; Sakai et al., 2000; Halmagyi et al., 2004), 고농도의 유리화 용액에서의 탈수에 의한 vitrification(Ahn and Skai, 1994; Martin and Gonzalez-Benito, 2005; Sakai et al., 2000; Halmagyi et al., 2004), 등 액체 질소(-196℃)의 초저온에 장기보존하는 방법이 시도되었으나, 아직 동결보존의 실용화 사례는 없는 것으로 알려져 있 다.Chrysanthemum is preserved as a nutrient resource in the field in consideration of genetic uniformity, so it may be lost due to physiological and pathological deterioration. To overcome this, slow-freezing by adjusting the cooling rate (Fukai, 1990, 1991; Halmagyi et al., 2004), encapsulation-dehydration by alginic acid coding and dehydration (Martin and Gonzalez-Benito, 2005; Sakai et al. , 2000; Halmagyi et al., 2004), vitrification by dehydration in high concentration vitrification solutions (Ahn and Skai, 1994; Martin and Gonzalez-Benito, 2005; Sakai et al., 2000; Halmagyi et al., 2004) Although long-term preservation of cryogenic liquid nitrogen (-196 ° C) has been attempted, there is no known practical application of cryopreservation.
본 발명은 국화와 같은 숙근 영양체 번식자원의 보존연구를 활성화하고, 기내 및 초저온동결보존 등 첨단 보존기술의 도입으로 동결장애 극복, 생존율 향상을 위한 제반 요인 탐색 및 최적조건 규명, 동결보존제의 생리적 영향 등 기초연구자료를 토대로 국화 특성에 따른 항구적인 보존 기술을 제공하고자 한다.The present invention activates conservation studies of trophic propagation resources such as chrysanthemums, and by introducing cutting-edge conservation technologies such as in-flight and cryogenic freezing preservation, explore the various factors for the improvement of survival rate, identify the optimal conditions, and find the physiological properties of cryopreservatives. Based on basic research data such as impact, we will provide permanent preservation technology according to chrysanthemum characteristics.
상기와 같은 과제를 해결하기 위하여 본 발명은 국화 유식물의 정아 또는 액아를 적출하고; 절편체를 0.3M-0.5M-0.7M 슈크로스 배지에서 3단계 전배양하며; 전배양된 시료를 로딩용액(17.5% 글리세롤+ 17.5% 슈크로스)에서 40~50분 배양하여 국화신초를 얻고; 국화신초를 얼음에 재운 A계열 용액에서 40-50분간 또는 실온의 B계열 용액에 60-90분간 쉐이킹하면서 탈수시킨 다음, 알루미늄 호일 위에 시료를 얹어서 액체 질소에 직접 급속동결하는 국화 신초의 초저온동결보존 방법을 제공한다. In order to solve the above problems, the present invention is to extract the chaff or liquid solution of chrysanthemum seedlings; Sections were precultured in three stages in 0.3M-0.5M-0.7M sucrose medium; Incubate the precultured sample for 40-50 minutes in a loading solution (17.5% glycerol + 17.5% sucrose) to obtain chrysanthemum buds; Ultra-low freeze preservation of chrysanthemum shoots, which are dehydrated while shaking chrysanthemum buds for 40-50 minutes in ice-based A series solution or 60-90 minutes in room temperature B series solution, and then placed on a sample of aluminum foil. Provide a method.
한편, 본 발명은 초저온동결된 국화 신초를 37-40℃ 슈크로스 용액(0.8M)에 직접 담가서 급속 해동하고, 해동된 국화 신초를 0.8M 슈크로스 용액에 언로딩시킨 후, 재생 배양용 배지에서 배양하는 초저온동결된 국화 신초의 재생 방법을 제공한다. Meanwhile, the present invention is rapidly thawed by directly immersing the cryogenic frozen chrysanthemum in 37-40 ° C. sucrose solution (0.8M), and unloading the thawed chrysanthemum in 0.8M sucrose solution, followed by regeneration culture medium. Provided is a method for regenerating cryogenic frozen chrysanthemum shoots.
본 발명은 액체 질소를 이용하여, 국화 등 영양체 유전자원을 액체 질소의 초저온에 동결보존하는 데 있어서 동결보존 후의 생존에 필수불가결한 동결보호제 유리화 용액과 로딩 용액을 개발하고, 이를 실재 국화 신초의 동결보존에 적용함으로써 장기보존할 수 있는 방법을 제공한다. The present invention develops a cryoprotectant vitrification solution and a loading solution which are indispensable for survival after cryopreservation in cryopreservation of nutrient gene sources such as chrysanthemum at cryogenic temperature of liquid nitrogen using liquid nitrogen, Applying to cryopreservation provides a method for long term preservation.
본 발명의 유리화 용액과 로딩 용액은 다양한 식물 종에 걸쳐서 광범위한 재료에 적용가능한 범용 동결보호제 혼합용액으로서 활용도가 높고, 국화에 적용한 작은 방울-유리화법에 의한 국화의 장기보존방법은 생존율과 재생률을 향상시킴으로써 포장에서 소실되기 쉬운 우량품종 등 국화와 같은 숙근류 유전자원을 장기보존함으로써 유전자원 및 종자산업분야에 널리 활용될 수 있을 것이다.The vitrification solution and the loading solution of the present invention are highly applicable as a general purpose cryoprotectant mixture solution applicable to a wide range of materials across various plant species, and the long-term preservation method of chrysanthemum by the small droplet-vitrification method applied to the chrysanthemum improves survival rate and regeneration rate. By long-term preservation of sperm root gene sources such as chrysanthemum, such as high-quality varieties that are easily lost in the packaging can be widely used in the genetic resources and seed industry.
실시예Example 1: 계대 1: passage 배양 기간에In the incubation period 따른 According 국화신초의Chrysanthemum 동결보존 전, 후의 생존율과 재생율 Survival and Regeneration Rates Before and After Cryopreservation
국화 기내 유식물 측아의 샘플링을 위한 최적 계대 배양 주기를 구명하고자 마디 배양기간에 따른 동결보존 전(-LN) 후(+LN)의 생존율(surv)과 재생률(rege)을 조사하였다. PVS3 유리화 용액에서 90분간 탈수시킨 탈수-무처리(도 2a, -LN)에서는 계대 배양 5.5-7주 처리구에서 생존율과 재생율이 가장 양호하였다. 동결보존 후에도 5.5주-7주에서 생존율(96.1%, 97.4%)과 재생률(88.5%, 91.4%)이 가장 높았다(도 2b). Survival (rev) and regeneration (pre-LN) before and after freezing preservation (-LN) were examined to determine the optimal passage period for sampling of seedlings in the chrysanthemum. In the dehydration-free treatment (FIG. 2A, -LN), which was dehydrated for 90 minutes in the PVS3 vitrification solution, the survival rate and regeneration rate were the best in the subculture 5.5-7 weeks. After cryopreservation, survival rates (96.1%, 97.4%) and regeneration (88.5%, 91.4%) were the highest at 5.5 to 7 weeks (Fig. 2b).
실시예Example 2: 2: PreculturePreculture 기간에 따른 Over time 국화신초의Chrysanthemum 동결보존 전, 후의 생존율과 재생율 Survival and Regeneration Rates Before and After Cryopreservation
국화 기내 유식물 측아의 전배양(pre-culture) 조건에 따른 동결보존 전(-LN) 후(+LN)의 생존율(surv)과 재생률(rege)을 조사하였다. 동결보존 후의 재생률은 0.3M-0.5M 또는 0.3M-0.5M sucrose의 2단계 전배양보다 0.3M-0.5M-0.7M sucrose 용액에서의 3단계 전배양이 높은 재생률을 보였으며, 각각 31-17-7시간 또는 31-17-17시간 배양할 경우 가장 높은 재생률을 보였다(전: 도 3a, 후: 도 3b). Survival (rev) and recovery (pre-LN) and pre-freezing (-LN) of pre-culture conditions of seedling side of chrysanthemum seedlings were investigated. Regeneration rate after cryopreservation showed higher regeneration rate in 0.3M-0.5M-0.7M sucrose solution than in two-step preculture of 0.3M-0.5M or 0.3M-0.5M sucrose, respectively 31-17 Incubation at -7 hours or 31-17-17 hours resulted in the highest regeneration rate (before: FIG. 3A, after: FIG. 3B).
실시예Example 3: 로딩 용액의 효과 3: effect of loading solution
이들 로딩 용액을 실재 국화 측아의 동결보존에 적용한 결과, 동결보존 전과 후 모두 PVS3의 35% 희석액인 C4(1.9M 글리세롤 +0.51M 슈크로스)가 가장 높은 생존율 및 재생률을 나타냈고, PVS3의 40% 희석액인 C6(2.17M 글리세롤 +0.58M 슈크로스)가 그 뒤를 이었다. 이들 용액은 글리세롤과 슈크로스가 동일 중량비를 이루고 있다(도 4a, 4b). Application of these loading solutions to cryopreservation of real chrysanthemum buds revealed that C4 (1.9M glycerol + 0.51M sucrose), 35% dilution of PVS3, before and after cryopreservation showed the highest survival and regeneration rate, and 40% of PVS3. The dilution C6 (2.17M glycerol + 0.58M sucrose) was followed. These solutions have the same weight ratio of glycerol and sucrose (FIGS. 4A and 4B).
이러한 결과를 활용하면 로딩 용액을 별도로 조제할 필요가 없이 PVS3 용액 을 35% 내외로 희석시켜서 사용함으로써 기존의 로딩 용액보다도 높은 재생률을 획득할 수 있다. By utilizing these results, it is possible to obtain a higher regeneration rate than the conventional loading solution by diluting the PVS3 solution to around 35% without having to prepare a loading solution separately.
실시예Example 4: 4: 유리화Vitrification 용액의 효과 Effect of solution
국화 신초에서 동결보존 전(도 5a)과 후(도 5b)의 생존율과 재생율은 유리화 용액의 농도와 조성에 따라 다양하였다. 유리화 용액들(PVS2와 변형용액들에 30분씩 또는 PVS3와 변형용액들에 150분씩)에 탈수시킨 국화 신초의 동결보존 전과 후의 생존율(surv)과 재생률(rege)은 도 5a, 5b와 같다. 고농도(32.5%-50.0%)의 슈크로스는 동결보존되었던 시료의 회복에 긍정적인 영향을 주었다. PVS3과 그 변형용액들에서 글리세롤과 슈크로스의 균형이 동결보존 시료의 재생에 긍정적인 영향을 주었고, 고농도의 글리세롤이 고농도의 슈크로스보다 더 효과적이었다. Survival and regeneration rates before and after cryopreservation (FIG. 5B) in chrysanthemum shoots varied with the concentration and composition of the vitrification solution. Survival and recovery rates before and after cryopreservation of chrysanthemum shoots dehydrated in vitrified solutions (30 minutes in PVS2 and modified solutions or 150 minutes in PVS3 and modified solutions) are shown in FIGS. 5A and 5B. High concentrations (32.5% -50.0%) of sucrose had a positive effect on the recovery of the cryopreserved sample. The balance of glycerol and sucrose in PVS3 and its modified solutions had a positive effect on the regeneration of cryopreserved samples, and higher concentrations of glycerol were more effective than high concentrations of sucrose.
실시예Example 5: 5: 유리화Vitrification 용액에서 탈수시간의 효과 Effect of Dehydration Time in Solution
A3 유리화 용액의 적정 탈수시간은 상온에서는 15-25분, 얼음에 재운 0℃에서는 40-55분이 적당하였고, B1 유리화 용액은 상온에서 60분이 적당하였다(도 6). The proper dehydration time of the A3 vitrification solution was 15-25 minutes at room temperature and 40-55 minutes at 0 ° C. on ice, and 60 minutes at room temperature for the B1 vitrification solution (FIG. 6).
[표1] 유리화 용액의 조성표Table 1 Composition of Vitrification Solution
※Con. : control 용액(기존 용액) ※ Con. : control solution (existing solution)
[표 2] 로딩 용액의 조성표Table 2 Composition of Loading Solution
※Con. : control 용액(기존 용액) ※ Con. : control solution (existing solution)
실시예Example 6: 6: 유리화Vitrification 용액에 따른 0.7M 0.7 M depending on the solution sucrosesucrose 전배양의Preculture 효과 effect
A계열의 유리화 용액(A1, A2)과 B계열의 유리화 용액(B1, B3)에 대해 각각 0.3M의 1단계 전배양체계(A)과 0.3M, 0.5M, 0.7M 슈크로스(sucrose) 배지에서 각각 하루, 일과시간, 아침까지 전배양하는 공정(B)을 적용한 경과, 동결보존 후(도 7b)의 재생율은 3단계 전배양체계를 적용한 B계열 용액에서 현저히 증가하였다(보존 전: 도 7a, 보존 후: 도 7b). 0.3M one-step preculture system (A) and 0.3M, 0.5M, 0.7M sucrose medium for A-type vitrification solution (A1, A2) and B-type vitrification solution (B1, B3), respectively The regeneration rate after the pre-cultivation process (B) and freeze-preservation (FIG. 7b) were significantly increased in the B-based solution applied with the three-stage pre-culture system (pre-preservation: Fig. 7a). After preservation: FIG. 7B).
도 1은 작은 방울-유리화법에 의한 국화 신초의 동결보존 과정 모식도이다. 1 is a schematic diagram of cryopreservation process of chrysanthemum shoots by small droplet-vitrification.
도 2는 계대 배양 기간에 따른 국화신초의 동결보존 전(도 2a), 후(도 2b)의 생존율과 재생률을 나타낸다.Figure 2 shows the survival rate and regeneration rate before and after cryopreservation of the chrysanthemum shoot (Fig. 2a), according to the passage culture period (Fig. 2b).
도 3은 Preculture 기간에 따른 국화신초의 동결보존 전(도 3a), 후(도 3b)의 생존율(surv)과 재생률(rege)을 나타낸다.Figure 3 shows the survival rate (surv) and regeneration (rege) before and after cryopreservation of the chrysanthemum buds according to the preculture period (Fig. 3a), (Fig. 3b).
도 4는 로딩 용액에 따른 국화 측아의 동결보존 전(LN-,도 4a)·후(LN+, 도 4b)의 생존율(surv) 및 재생률(rege)을 나타낸다.FIG. 4 shows the survival and regeneration of pre-Lyopreservation (LN-, FIG. 4A) and post-LN +, FIG. 4B of chrysanthemum buds with loading solution.
도 5는 유리화 용액들(A계열(PVS2와 변형용액들)에 30분씩 또는 B계열(PVS3와 변형용액들)에 150분씩)에 탈수시킨 국화 신초의 동결보존 전(도 5a)과 후(도 5b)의 생존율(surv)과 재생률(rege)을 나타낸다.5 shows before and after cryopreservation of the chrysanthemum shoots dehydrated in vitrification solutions (30 minutes in series A (PVS2 and modified solutions) or 150 minutes in series B (PVS3 and modified solutions)). Survival and rege of 5b) are shown.
도 6은 유리화 용액들의 탈수시간에 따른 국화 신초의 동결보존 전(도 6a)과 후(도 6b)의 생존율(surv)과 재생률(rege)을 나타낸다.FIG. 6 shows the survival and regeneration rates before and after cryopreservation (FIG. 6A) and FIG. 6B of chrysanthemum shoots with dehydration time of vitrification solutions.
도 7은 유리화 용액과 0.7M sucrose 전배양에 따른 국화 신초의 동결보존 전(도 7a)과 후(도 7b)의 생존율(surv)과 재생률(rege)을 나타낸다.Figure 7 shows the survival (surv) and recovery (rege) before and after cryopreservation (Fig. 7a) and (7b) of chrysanthemum shoots according to the vitrification solution and 0.7M sucrose preculture.
도 8은 국화 신초의 동결보존 전후의 유식물체 사진이다(도 8a: 마디 배양, 도 8b: 동결보존 후 재생 배양 3주, 도 8c: 동결보존 후 재생 배양 8주). Figure 8 is a picture of the seedlings before and after cryopreservation of chrysanthemum shoots (Fig. 8a: node culture, Figure 8b: three weeks of regeneration culture after cryopreservation, Figure 8c: 8 weeks of regeneration culture after cryopreservation).
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102550417A (en) * | 2012-01-12 | 2012-07-11 | 天津中植科生物技术有限公司 | Cultivation of Hangzhou white chrysanthemum polyploidy good varieties and one-step seedling rapid propagation technology |
| CN103828710A (en) * | 2014-03-18 | 2014-06-04 | 南京农业大学 | Efficient chrysanthemum cross-breeding method |
| CN105475133A (en) * | 2015-11-30 | 2016-04-13 | 华南农业大学 | Cryopreservation method for plant germplasm of Eriobotrya by using combined method of programmed freezing method and vitrification method |
| CN111513061A (en) * | 2020-05-22 | 2020-08-11 | 上海市农业生物基因中心 | Ultralow-temperature preservation and recovery culture method for alum root clump buds |
| CN111616140A (en) * | 2020-06-11 | 2020-09-04 | 中国农业科学院特产研究所 | Vitrification ultralow-temperature preservation method and application of actinidia arguta dormant bud |
| CN111685109A (en) * | 2020-07-01 | 2020-09-22 | 山东省林木种质资源中心 | Ultralow-temperature preservation and recovery culture method for Chinese chestnut hypocotyls |
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2009
- 2009-04-03 KR KR1020090028899A patent/KR20100110525A/en not_active Application Discontinuation
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102550417A (en) * | 2012-01-12 | 2012-07-11 | 天津中植科生物技术有限公司 | Cultivation of Hangzhou white chrysanthemum polyploidy good varieties and one-step seedling rapid propagation technology |
| CN103828710A (en) * | 2014-03-18 | 2014-06-04 | 南京农业大学 | Efficient chrysanthemum cross-breeding method |
| CN105475133A (en) * | 2015-11-30 | 2016-04-13 | 华南农业大学 | Cryopreservation method for plant germplasm of Eriobotrya by using combined method of programmed freezing method and vitrification method |
| CN111513061A (en) * | 2020-05-22 | 2020-08-11 | 上海市农业生物基因中心 | Ultralow-temperature preservation and recovery culture method for alum root clump buds |
| CN111513061B (en) * | 2020-05-22 | 2022-05-03 | 上海市农业生物基因中心 | Ultralow-temperature preservation and recovery culture method for alum root clump buds |
| CN111616140A (en) * | 2020-06-11 | 2020-09-04 | 中国农业科学院特产研究所 | Vitrification ultralow-temperature preservation method and application of actinidia arguta dormant bud |
| CN111685109A (en) * | 2020-07-01 | 2020-09-22 | 山东省林木种质资源中心 | Ultralow-temperature preservation and recovery culture method for Chinese chestnut hypocotyls |
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| Gupta et al. | Cryopreservation of apple (Malus domestica'Benoni') dormant buds using two-step freezing method | |
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